Fluid Secretion

Millions of Americans (and more around the world) have reduced fluid secretion of their salivary glands.  This condition of "dry mouth" produces an enhanced risk of oral and systemic disease and leads to a deterioration in the ability to speak, chew, and swallow.  We are involved in a highly cooperative research program (with James Melvin, Trevor Shuttleworth, Dave Yule) that aims to understand the molecular basis for fluid secretion in salivary glands and to determine the causes of certain types of "dry mouth".

The trigger for salivary gland fluid secretion is activity of the parasympathetic nerve innervating the glands.  Action potentials in this nerve release acetylcholine which causes an increase in Ca²⁺ ions inside the acinar cells.   Increased intracellular Ca²⁺ activates K and Cl ion channels and the movement of these ions bring along the water that constitutes the fluid secretion.

Thus, our goal is to identify the Ca²⁺-activated K channels in parotid acinar cells and determine how their specific properties underlie their role in fluid secretion.  We have identified two types of Ca²⁺-activated K channels in parotid acinar cells (Nehrke et al., 2003- see Publications).  These two channels differ in their Ca²⁺ sensitivity, single channel conductance, and pharmacology.  One of these has a single channel conductance near 20 pS and the other a conductance near 150 pS.  Other Ca²⁺-activated K channels (not expressed in parotid cells) has a smaller conductance near 8 pS.  These three classes of Ca²⁺-activated K channels have been called SK, IK, and maxi-K for "small", "intermediate" and "maximal" conductance K channels.

Since we found  intermediate-conductance and  large-conductance Ca²⁺-activated K channels in parotid acinar cells, we tested for the expression of these specific genes in.  We found that messenger RNA (mRNA) from the IK1 gene (also known as Kcnn4 or K_Ca3.1) can be visualized by in situ hybridization and Northern blot analysis (see figure).  We have found that the properties of heterologously expressed mIK1closely matches one of the native parotid channels. 

The maxi-K channel in parotid acinar cells looks to be a special mouse splice variant of the Slo (or Kcnma1 or K_Ca1.1) gene that is, co-incidently, almost identical to the most common human splice variant of the gene.  In addition, parotid glands express the also express the b4 subunit partner of maxi-K channels.  The properties of heterologously expressed mSlo + b4 match those of the maxi-K Ca²⁺-activated K channel in parotid acinar cells.

Thus, it appears that salivary gland fluid secretion depends on the activity of two types of Ca²⁺-activated K channels and these are likely IK1 and Slo + b4 but it is important to verify these identifications  through the use of gene "knock-out" mice.  Keith Nehrke and Catherine Ovitt in Jim Melvin's lab have deleted the Kcnn4 (IK1) gene in a mouse strain and we have verified that this gene encodes the intermediate conductance, IK1 K channel since the knock-out mice lack this current (Begenisich et al., 2004). 

 We have recently obtained Slo-null mice from Rick Aldrich's lab and verified the absence of the maxi-K channel in parotid acinar cells.  We are currently investigating the physiological properties of parotid acinar cells from these Slo-null mice.

Having established the genes that encode the two Ca²⁺-activated K channels expressed in parotid glands, our attention has turned to determining the specific roles for these two channels: why are they BOTH expressed?  "What does each one do?"  The focus on these questions was sharpened by our finding that both IK1-null  and Slo-null mice have perfectly normal fluid secretion (as least as measured with a standard, simple secretion protocol) but (after cross-breeding) animals deficient in both K channels had severely impaired fluid secretion.   It may be that these channels are used for different aspects of fluid secretion- perhaps one for basal or low levels and the other for high levels of fluid secretion.  Answering these next set of questions will require determining the Ca²⁺ sensitivity of these two channels in parotid acinar cells and developing new protocols for testing fluid secretion.

A Parotid Gland

The parotid gland is the most important contributor to salivary fluid secretion.  It consists of clumps of acinar cells (shown here) connected by fluid transporting ducts.  Picture courtesy of Dave Yule and Jason Bruce.

Ca²⁺ oscillations in a parotid acinar cell

Oscillations of intracellular Ca induced by carbachol (CCh) a mimic of the physiological transmitter, acteylcholine.  Courtesy of Dave Yule and Jason Bruce.

IK1 expression in parotid acinar cells